Contrast agents

ABSTRACT

Contrast agents comprising microbubble-generating carbohydrate microparticles having a surfactant admixed within the microparticulate structure, with the proviso that the surfactant is not a C 10-20  fatty acid, are disclosed. Processes for preparing contrast agents also are disclosed.

This invention relates to novel contrast agents, more particularly tonew microparticulate contrast agents of use in diagnostic imaging.

It is well known that ultrasonic imaging comprises a potentiallyvaluable diagnostic tool, for example in studies of the vascular system,particularly in cardiography, and of tissue microvasculature. A varietyof contrast agents has been proposed to enhance the acoustic images soobtained, including suspensions of solid particles, emulsified liquiddroplets, gas microbubbles and encapsulated gases or liquids. It isgenerally accepted that low density contrast agents which are easilycompressible are particularly efficient in terms of the acousticbackscatter they generate, and considerable interest has therefore beenshown in the preparation of gas-containing and gas-generating systems.

Initial studies involving free gas microbubbles generated in vivo byintracardiac injection of physiologically acceptable substances havedemonstrated the potential efficiency of such bubbles as contrast agentsin echocardiography; such techniques are severely limited in practice,however, by the short lifetime of the free bubbles. Interest hasaccordingly been shown in methods of generating and/or stabilising gasmicrobubbles for echocardiography and other ultrasonic studies, forexample using emulsifiers, oils, thickeners or sugars.

Techniques involving the use of sugars in ultrasound contrast agents aredescribed in, for example, U.S. Pat. No. 4,681,119, U.S. Pat. No.4,442,843 and U.S. Pat. No. 4,657,756, which disclose the use ofparticulate solids having a plurality of gas-filled voids and preferablyalso a plurality, of nuclei for microbubble formation. EP-A-0123235 andEP-A-0122624 suggest ultrasound contrast agents consisting ofsurfactant-coated or surfactant-containing gas-containing microparticleswhich may include a variety of sugars. Where surfactant-containingmicroparticles are described, these are prepared simply by comminglingthe surfactant with the microparticulate materials, e.g. by trituration.

DE-A-3834705 proposes the use of suspensions containing microparticlesof mixtures of at least one C₁₀₋₂₀ fatty acid with at least onenon-surface active substance, including sugars such as cyclodextrins,monosaccharides, disaccharides or trisaccharides, as well as otherpolyols and inorganic and organic salts; in practice only the use ofgalactose as the non-surface active material and only the use ofsaturated fatty acids are exemplified. The microparticulate materialsare typically prepared by coprecipitating the fatty acid and non-surfaceactive substance and comminuting the resulting product, e.g. using anair-jet mill.

One material of, the type described in DE-A-3834705, SHU 508 (Levovist6), is described in the following publications: Schlief, R. et al.,Circulation Supplement III (1990) 82, p. 28; Schartl, M. et al.,Circulation Supplement III (1990) 82, p. 261; Fritzsch, T. et al.,Invest. Radiol. (1990) 25 (Suppl), pp. 160-161; Schlief, R. et al.,Echocardiography (1990) 7, pp. 61-64; Loughery, E. J. et al.,Echocardiography (1990) 7, pp. 279-292; and Smith, M. D. et al., JACC(1989) 13, pp. 1622-1628.

Gas-containing contrast media are also known to be effective in magneticresonance. (MR) imaging, e.g. as susceptibility contrast agents whichwill act to reduce MR signal intensity oxygen-containing contrast mediaalso represent potentially useful paramagnetic MR contrast agents.

Furthermore, in the field of X-ray imaging it has been observed thatgases such as carbon dioxide may be used as negative oral contrastagents.

A general disadvantage of most of the existinggas-containing/gas-generating particulate contrast agents such as thesugar-based agents discussed above is their relative lack of stabilityin vivo. This is a particular problem in applications such asechocardiography, where there is a need for improved contrast agentscombining sufficient stability and small microbubble size (typicallyless than about 10 μm, preferably less than about 7 μm) to permitpassage through the pulmonary capillary bed and so allow enhancedvisualisation of the left side of the heart, preferably for more thanone passage of circulation. There is accordingly a need for contrastagents which generate microbubble systems exhibiting good stabilitywhile still providing an effective level of contrast efficiency.

The present invention is based on our finding that contrast agentscomprising microparticles of a carbohydrate having a surfactant admixedtherewith (but excluding the previously disclosed mixtures of galactoseand saturated C₁₀₋₂₀ fatty acids) may be used to generate microbubblesystems exhibiting enhanced contrast effect and/or stability relative topreviously proposed carbohydrate-based contrast agents. In theultrasound field this may be demonstrated by, for example, in vitromeasurements of initial attenuation levels and the half lives of theattenuative effect; a useful indication of the combined effect of theseproperties is the integral obtained by determining the area under thecurve of a plot of attenuation against time.

The term “surfactant” as used herein means any compound havingamphiphilic properties capable of modifying surface tension.

Thus, according to one aspect of the present invention, there areprovided contrast agents comprising microbubble-generating carbohydratemicroparticles having a surfactant admixed within the microparticulatestructure, with the proviso that the surfactant is not-a saturatedC₁₀₋₂₀ fatty acid when the microparticulate carbohydrate is galactose.

The microparticulate carbohydrate is preferably water soluble, andsubject to the foregoing proviso may for example be selected fromhexoses such as glucose, fructose or galactose; disaccharides such assucrose, lactose or maltose; pentoses such as arabinose, xylose orribose; and polysaccharides such as α-, β- and δ-cyclodextrins,maltodextrin and glycogen; the term “carbohydrate” as used herein isalso intended to embrace sugar alcohols, e.g. alditols such as mannitolor sorbitol. Microparticles of the above carbohydrates will normallyhave gas present as an inclusion in the voids of their crystal structureand/or adhered to their surface, which gas may generate microbubbleswhen, for example, the microparticles are suspended or dissolved in aninjectable carrier liquid, for example water for injection, an aqueoussolution of one or more inorganic salts (e.g. physiological saline-or aphysiological buffer solution), an aqueous solution of a monosaccharide(e.g. glucose or galactose) or disaccharide (e.g. lactose), or anaqueous solution of a physiologically tolerable monohydric or polyhydricalcohol (e.g. ethanol, propanol., isopropanol, ethylene glycol,propylene glycol, glycerine or polyethylene glycol).

In addition to or alternatively to air, any biocompatible gas may beemployed in the contrast agents of the invention, for example nitrogen,oxygen, hydrogen, nitrous oxide, carbon dioxide, helium, argon, sulphurhexafluoride and low molecular weight optionally fluorinatedhydrocarbons such as methane, acetylene or carbon tetrafluoride. Theterm “gas” as used herein includes any substance in the gaseous form at37° C. The gas may be contained in the contrast agent in such a way thatbefore use the product is non-contrast giving but becomes-effective onadministration, e.g. as a result of the gas forming microbubbles as asoluble carbohydrate matrix dissolves.

Additionally or alternatively the carbohydrate may incorporate one ormore gas precursors, including carbonates and bicarbonates (e.g. sodiumor ammonium bicarbonate) and aminomalonate esters.

Subject to the foregoing proviso a wide variety of surfactants may beused in the ultrasound contrast agents of the invention; it will ofcourse be appreciated that the surfactant is required to bebiocompatible, i.e. that it should be physiologically tolerable in thequantities in which it is to be administered. The surfactant isadvantageously biodegradable in vivo or otherwise readily eliminablefrom the system.

The surfactant may, for example, be an amphiphilic lipid, e.g. selectedfrom fatty acids and salts (e.g. alkali metal salts) thereof, steroidacids, sterols, phospholipids and glycolipids. Such lipids include highmolecular weight (e.g. C₁₀₋₅₀) straight chain saturated and unsaturatedaliphatic acids, such as capric, palmitic, hexadecanedioic, stearic,linolenic, behenic, docosanedioic and melissic acids; aralkanoic acids,e.g. phenyl lower alkanoic acids such as. 2-phenylbutyric acid; salts ofany of the foregoing acids; mono- and di-glycerides, for exampleglyceryl esters of high molecular weight (e.g. C₁₀₋₅₀) aliphatic acids,such as glyceryl monolaurate; cholanic acids such as 50-cholanic acid;cholesterol; sorbitan esters of fatty acids such as Span-type materials;high molecular weight (e.g. C₁₀₋₅₀) straight chain aliphatic alcoholssuch as stearyl alcohol and cetyl alcohol; phospholipids such asphosphatidyl choline (lecithin) and dioleoylphosphatidyl ethanolamine(DOPE); and mixtures thereof.

Other surfactants which may be employed include anionic surfactants, forexample alkali metal alkyl sulphates such as sodium lauryl sulphate andsulphonated esters such as sodium dioctyl sulphosuccinate (docusate);and non-ionic surfactants, for example polyoxyethylene-polyoxyproplyenecopolymers (e.g. poloxamers such as Pluronic F68) and polyoxyethylatedsorbitan esters (e.g. polysorbates such as Tween-type materials).

The surfactant moiety may if desired be covalently linked to a substratesuch as a carbohydrate prior to its admixture with the principalmicroparticulate carbohydrate. Thus, for example, a fatty acid such aspalmitic acid (preferably in the form of a reactive derivative such as acorresponding acyl halide) may be used to esterify a (preferablyappropriately O-protected) sugar such as galactose and the resultinglipophilically modified carbohydrate used as the 0.20 surfactant inaccordance with the invention.

The surfactant may, for example, be present in an amount of 0.01-5.0 wt.%, preferably 0.1-2.0 wt. %, relative to the microparticulatecarbohydrate.

The contrast agents of the invention may be used in a variety ofdiagnostic imaging techniques, including ultrasound, MR and X-rayimaging. Their uses in diagnostic ultrasonic imaging and MR imaging,e.g. as susceptibility contrast agents, constitute preferred features ofthe invention.

The contrast agents of the invention may be prepared by any convenientmethod which leads to physical admixture of the surfactant within themicroparticulate structure of the carbohydrate and to production ofmicroparticles of the desired size.

In one preferred method according to the invention the carbohydrate andthe surfactant are each dissolved in appropriate mutually misciblesolvents (e.g. water in the case or the carbohydrate and a lower alkanolsuch as ethanol in the case of lipid surfactants such as fatty acids),the resulting solutions are mixed, the solvents are removed (e.g. byevaporation under reduced pressure), and the resulting solid mixture ismicronised to yield the desired microparticles. It will be appreciatedthat all such operations should be effected under sterile conditions.

In an alternative method according to the invention a (preferablyaqueous) solution of the carbohydrate is mixed with a liposome-formingmaterial (e.g. a thin film of a lipid such as lecithin formed on theinner surface of the mixing vessel by evaporating the solvent from asolution of the lipid in an appropriate organic solvent, for example achlorinated hydrocarbon such as chloroform) so as to form aliposome-containing carbohydrate solution from which the solvent may beremoved (e.g. by freeze-drying) to yield a product comprisingcarbohydrate-containing liposomes; this product may be micronised togiven microparticles of the desired size.

In general conventional micronisation techniques such as grinding ormilling may be employed in processes according to the invention.Ball-milling of the solid mixture has been found to be particularlyadvantageous, permitting the preparation of microparticles in the formof aggregates (for example having an aggregate size of 20-125micrometres, such as 30-50 micrometres) of particles having a particlesize of, for example, 1-50 micrometres, such as 1-10 micrometres. Suchaggregates will tend to contain a substantial volume of air adsorbed ontheir surfaces and entrained in voids such as interparticle cavities orat grain boundaries between the crystallites. The particle size may, forexample, be selected to be substantially commensurate with the desiredmicrobubble size. In ultrasonic applications such as echocardiography,in order to permit free passage through the pulmonary system and toachieve resonance with the preferred imaging frequencies of about 0.1-15MHz, it may be convenient to employ microbubbles and microparticleshaving an average size of 0.1-10 μm, e.g. 1-7 μm; the use ofmicroparticles of average size 1-4 μm to generate microbubbles with anaverage size of 4-7 μm is generally advantageous. Substantially largerbubbles and particles, e.g. with average sizes up to 500 μm, may howeverbe useful in other applications, for example gastrointestinal imaging.

Ultrasound contrast agents in the form of microparticles comprising amicrobubble-generating carbohydrate in admixture with an amphiphilicorganic acid containing in excess of 20 carbon atoms are the subjectmatter of our international patent application cofiled herewith andclaiming priority from British patent application No. 9200387.0.

The following non-limitative Examples serve to illustrate theinvention:—

EXAMPLES 1-18

General Procedure

D-(+)-galactose (10.0 g) was dissolved in distilled water (14.2 g) at50° C., sterile filtered and cooled on ice to a temperature of 4-8° C.The stated amounts of the surfactants (in % w/w relative to thegalactose) listed in Table I were each dissolved in the amount of 96%ethanol (or water in Examples 5 and 6) shown in the Table, at 50-78° C.,and the resulting solution was sterile filtered and then asepticallyadded to the cold aqueous galactose solution under stirring. Theresulting mixture was evaporated to dryness under reduced pressure (10torr, 40° C.), and the resulting solid product was dried in a desiccatorovernight and then ground for 10 minutes under aseptic conditions in astainless steel ball mill having a 50 ml grinding cup and 3×20 mm balls(fetsch centrifugal ball mill, S1) The ground product was dried in adesiccator for 24 hours. TABLE I Amount of Amount of ethanol ExampleSurfactant (or water) No. Surfactant (% w/w) (g) 1 Lecithin 1.0 1.2 2 ″0.2 1.2 3 Sodium Lauryl Sulphate 1.0 1.0 (water) 4 ″ 0.1 1.0 (water) 5Span 80 1.0 1.2 6 ″ 0.1 1.2 7 Span 85 1.0 1.2 8 ″ 0.1 1.2 9 Pluronic F681.0 1.2 10 ″ 0.1 1.2 11 Sodium Docusate 1.0 1.2 12 ″ 0.1 1.2 13 DOPE 1.01.2 14 ″ 0.1 1.2 15 α-Glyceryl Monolaurate 0.2 3.2 Glyceryl Tripalmitate0.2 Cholesterol 0.2 Cholesterol Acetate 0.2 Cholesterol Benzoate 0.2 16α-Glyceryl Monolaurate 0.02 1.2 Glyceryl Tripalmitate 0.02 Cholesterol0.02 Cholesterol Acetate 0.02 Cholesterol Benzoate 0.02 17Hexadecanedioic Acid 0.2 1.2 18 Linolenic Acid 1.0 1.2

EXAMPLES 19-22

The general procedure for Examples 1-18 was repeated except that theD-(+)-galactose was replaced by the carbohydrates listed in Table II, inthe amounts and using the quantities of water shown, and that thesurfactant used was palmitic acid (0.2% w/w relative to thecarbohydrate) dissolved in 96% ethanol (1.2 g). TABLE II Amount ofAmount of Example Microbubble-generating Carbohydrate water No.Carbohydrate (g) (g) 19 Xylose (BDH) 10.0 14.2 20 Maltodextrin 10.0 14.221 Glycogen (Merck) 5.0 17.2 22 α-Cyclodextrin (Sigma) 5.0 12.2

EXAMPLE 23

6-O-Palmitoyl-b-galactopyranose/Galactose Mixtures

(A) 6-O-Palmitoyl-1,2,3,4-diisopropylidene-D-galactopyranose

1,2,3,4-Diisopropylidene-D-galactopyranose (Sigma, 13.4 g, 51.3 mmol)and triethylamine (7.15 ml, 51.3 mmol) were dissolved in methylenechloride (150 ml) and cooled to 0° C. Palmitoyl chloride (Aldrich, 14.1g, 51.3 mmol) dissolved in methylene chloride (100 ml) was addeddropwise with: stirring over 1 h. The cooling bath was removed and thereaction mixture was stirred overnight. Precipitated triethylaminehydrochloride was removed by filtration, the filtrate was transferred toa separating funnel and extracted with water (3×50 ml), dried over MgSO₄and the solvent was removed in vacuo. The residue was a light brownishoil which solidified to waxy crystals. Crude yield: 23 g. The crudeproduct was used without further purification. A small aliquot wasrecrystallized for characterisation. FT-IR:CO-1734 cm⁻¹. ¹³C-NMR:CO-ester 172.79. Mp. 124-127° C.

(B) 6-O-Palmitoyl-D-galactopyranose

6-O-Palmitoyl-1,2,3,4-diisopropylidene-D-galactopyranose (6 g) wasdissolved in acetic acid (25 ml) and heated to 100° C. under nitrogenfor 6 h. During subsequent cooling to room temperature, the productprecipitated from the solvent, and was left at room temperatureovernight. The crystals were collected by filtration and dried undervacuum. Yield: 3.3 g. The product was characterized by FT-IR:CO-1734cm⁻¹; OH-3464 cm¹.

(C) 6-O-Palmitoyl-D-galactopyranose/Galactose Mixtures

(i) D-(+)-galactose (2 g) was dissolved in purified water (2.87 g) andsterile filtered. 6-O-Palmitoyl-D-galactopyranose (0.25 g) prepared asdescribed in (B) above was dissolved in ethanol (3 g) and sterilefiltered. The solution of the palmitoyl-galactopyranose was added to thegalactose solution under stirring and the whole mixture was taken todryness under vacuum (10 torr, 50° C.). The product was dried in adesiccator overnight.

(ii) The procedure of (i) was repeated using6-O-palmitoyl-D-galactopyranose (0.50 g) dissolved in ethanol (6 g).

EXAMPLE 24

Freeze-Dried Liposomes Containing D-(+)-Galactose Particles

1 ml 100 mg/ml phosphatidylcholine was dissolved in 10 ml chloroform.The mixture was poured into a round bottom flask, and the organic phasewas evaporated at 40° C. in such a way that a thin film of thephosphatidylcholine was formed on the inner surface of the flask. 10 mlof a sterile, pyrogen free 40% aqueous D-(+)-galactose solution was thenadded at 40° C. and the flask was kept rotating for 1 hour. The aqueoussolution containing liposomes and dissolved galactose was thenfreeze-dried for 24 hours, and the resulting product consisting offreeze-dried-galactose and freeze-0.5 dried galactose-filled liposomeswas then ground in a ball-mill to yield a product with a particle sizedistribution of 1-20 μm.

EXAMPLE 25

Echogenicity In Vitro

10 ml of propylene glycol mixed with 90 ml of 5% dextrose in water wasused as a carrier liquid for determining the echogenicity of productsaccording to the Examples. 11.0 g of each product to be tested wasdispersed in 3.0 ml of the carrier liquid and shaken for seconds. Theresulting mixture was added to 52 ml of 5% human serum albumin infusionsolution in the measurement cell and the acoustic effects of theproducts were investigated by measuring the acoustic transmissionthrough the samples using a 5 MHz broadband transducer in apulse-reflection technique. The temperature in the measurement cell wasstabilised to 37° C. and circulation of the liquid was maintained bymeans of stirring at a constant rate. Ultrasound transmission throughthe samples was measured as a function of time over a duration of 390seconds. Results were normalized to measurements on a referenceconsisting of 55 ml of 5% human serum albumin infusion solution.

Results for representative exemplified products and comparative resultsfor unmodified milled D-(+)-galactose are shown in the accompanyingdrawing as FIG. 1 it will be apparent that these products exhibit astrong effect on ultrasonic attenuation in vitro, an effect whichpersisted for several minutes.

1-13. (canceled).
 14. The method of generating an enhancedechocardiographic image of a human or non-human body comprising:administering into the pulmonary system of said body anechocardiographic contrast enhancing amount of a contrast agentcomprising gasmicrobubble-generating aggregates of microparticles;applying to a part of said body ultrasound at a frequency of 0.1 to 15MHZ; and generating said image; said microparticles comprising a watersoluble matrix material and a surfactant, the microbubbles generated bysaid aggregates comprising SF₆ or a fluorinated low molecular weighthydrocarbon, said aggregates being 20-125 μm in size and saidmicroparticles having an average size of 0.1 to 50 μm.
 15. The method asclaimed in claim 14 in which the surfactant is selected from the groupconsisting of straight chain aliphatic carboxylic acids and salts,sorbitan esters and mono- and di-glycerides thereof; aralkanoic acidsand the salts thereof; steroid acids; sterols; straight chain aliphaticalcohols; phospholipids; alkali metal alkyl sulphates and sulphonatedesters; polyoxyethylene-polyoxypropylene copolymers; polyoxyethylatedsorbitan esters; and mixtures of any of the foregoing.
 16. The method asclaimed in claim 14 in which the surfactant comprises a lipophilicallymodified carbohydrate.
 17. The method as claimed in claim 14 in whichthe surfactant is present in an amount of 0.1-2.0% w/w relative to thewater soluble matrix.
 18. The method as claimed in claim 14 for whichthe microbubbles generated by said aggregates contain air in admixturewith said SF₆ or fluorinated hydrocarbon.
 19. The method as claimed inclaim 14 for which the microbubbles generated by said aggregatescomprise carbon tetrafluoride.
 20. The method as claimed in claim 14 inwhich the water soluble matrix is a carbohydrate.
 21. The method asclaimed in claim 20 in which the carbohydrate is a polysaccharide. 22.The method as claimed in claim 20 in which the carbohydrate is a sugaralcohol.
 23. The method as claimed in claim 14 which is non-contrastgiving before use, but which becomes effective on administration. 24.The method as claimed in claim 15 which is non-contrast giving beforeuse, but which becomes effective on administration.
 25. The method asclaimed in claim 16 which is non-contrast giving before use, but whichbecomes effective on administration.
 26. The method as claimed in claim17 which is non-contrast giving before use, but which becomes effectiveon administration.
 27. The method as claimed in claim 18 which isnon-contrast giving before use, but which becomes effective onadministration.
 28. The method as claimed in claim 19 which isnon-contrast giving before use, but which becomes effective onadministration.